Chapter 12: Self-contained Biocatalysts

Self-sufficient biocatalysts encompass all necessary catalytic activities to perform the reaction of interest. In practice, this nearly always means providing enzymes to regenerate redox cofactors, especially NAD(P)⁺ and NAD(P)H. There have been two basic approaches to making biocatalytic reactions self-sufficient: chimeric enzymes and co-expressed enzymes. Like their mythological counterparts, biocatalytic chimeras fuse one or more independent proteins into a single covalent assembly. This approach has been particularly popular for cytochrome P450's, as these systems require three components that are often found in three different proteins in Nature. In addition to P450's, several other recent examples of chimeric biocatalysts are described. Enzyme co-expression relies on multiple proteins, but present at high levels in a single cell (usually Escherichia coli). Cofactor supply to dehydrogenases (amino acid and alcohol) has commonly used this approach, and a number of recent examples are described. In addition, the use of co-expressed enzymes to supply nicotinamides in their oxidized form (for substrate oxidations) is discussed. Finally, two examples of self-sufficient biocatalysts using co-expression, but in non-E. coli hosts, are covered.